Zearalenone detoxification compositions and methods

ABSTRACT

The present invention provides a bacterial microorganism of the Rhodococcus or Nocardia species having the ability to degrade zearalenone.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of application Ser. No. 08/753,316,filed Nov. 22, 1996, now U.S. Pat. No. 5,846,812.

TECHNICAL FIELD

The present invention relates generally to the detection and isolationof zearalenone-degrading organisms and to compositions and methods forthe detoxification or degradation of zearalenone. This method has broadapplication in agricultural biotechnology and crop agriculture and inthe improvement of food grain quality.

BACKGROUND OF THE INVENTION

Fungal diseases are common problems in crop agriculture. Many strideshave been made against plant diseases as exemplified by the use ofhybrid plants, pesticides and unproved agricultural practices. However,as any grower or home gardener can attest, the problems of fungal plantdisease continue to cause difficulties in plant cultivation. Thus, thereis a continuing need for new methods and materials for solving theproblems caused by fungal diseases of plants. These problems can be metthrough a variety of approaches. For example, the infectious organismscan be controlled through the use of agents that are selectivelybiocidal for the pathogens. Another method is interference with themechanism by which the pathogen invades the host crop plant. Yet anothermethod, in the case of pathogens that cause crop losses, is interferencewith the mechanism by which the pathogen causes injury to the host cropplant. Still another method, in the case of pathogens that producetoxins that are undesirable to mammals or other animals that feed on thecrop plants, is interference with toxin production, storage, oractivity.

Within the Fusarium sp are several important pathogens of corn and othercereals in various countries. In corn, Fusarium is known to cause root,stem and ear rot that results in severe crop reduction. The etiology ofFusarium ear mold is poorly understood, although physical damage to theear and certain environmental conditions can contribute to itsoccurrence(Nelson PE (1992) "Taxonomy and Biology of Fusariummoniliforme." Mycopathologia 117: 29-36). Fusarium may be isolated frommost field grown maize, when no visible mold is present. Therelationship between seedling infection and the stalk and ear diseasescaused by Fusarium is not clear. Genetic resistance to visible kernelmold has been identified.(Gendloff E, Rossman E, Casale W, Isleib T,Hart P, 1986, "Components of resistance to Fusarium ear rot in fieldcorn." Phytopathology 76: 684-688; Holley RN, Hamilton PB, Goodman MM,1989, "Evaluation of tropical maize germplasm for resistance to kernelcolonization by Fusarium moniliforme." Plant Dis 73: 578-580). Themycotoxins produced by the Fusarium species that infect plants mayaccumulate in infected plants or in stored grains, presenting serioushealth consequences for livestock, humans, and other consumers of meator other food products of such livestock. Fusarium infection has beenassociated with chronic or acute mycotoxicoses in both farm animals andman (Botallico, et al.). An important mycotoxin that has been found tobe produced by certain Fusarium sp. and has been identified inFusarium-infected crops is zearalenone.

Zearalenone, produced mainly by Fusarium graminearum (perfect form isGibberella zeae), occurs in Fusarium-infected corn and to a lesserextent in other starchy cereal seeds. Zearalenone has been detected inhay, feed, corn, sorghum, dairy rations and barley that caused toxicosisin livestock in various countries (Ueno, et al. CRC Critical Rev.Toxicol. 14:99, 1985). When consumed by swine, it may incite anestrogenic response, including infertility, reduced litter size and weakpiglets (Mirocha, 1971). Zearalenone has also been shown to causeabortion, vomiting and diarrhea in animals that consume the mycotoxin(Kollarczik, 1994, Nat. Toxins 2:105). It is also physiologically activein cattle, rats, mice, guinea pigs, poultry and plants (Mirocha, 1971;Stob, 1992). In rats, it has been shown to be teratogenic (Ueno et al.,Cancer Res. 36:445, 1976; Ueno et al., Cancer Res. 38:536, 1978).Zearalenone has also been shown to induce modulation of uterine tissuesin mice (Ueno, et al. Jap. J. Exp. Med. 45:199, 1970).

There is a need in the art for novel methods with which zearalenone maybe eliminated from a plant or harvested grain. It is consideredimportant by those skilled in the art to continue to develop inventionsin order to protect the final consumer of a plant or harvested grain.The present invention provides the reagents and methodologies necessaryto ameliorate plants and harvested grains from zearalenone.

SUMMARY OF THE INVENTION

In one embodiment, the present invention provides a wild-type organismhaving the ability to degrade or detoxify zearalenone or structurallyrelated mycotoxins. The present invention may further include a mutantof the wild-type organism that has the ability to degrade or detoxifyzearalenone or structurally related mycotoxins. The present inventionalso provides a method for the isolation and utilization of azearalenone-degradation gene encoding a gene product having the abilityto degrade or detoxify zearalenone or structurally related mycotoxins.In another embodiment, the present invention provides for the generationof transformants into which the zearalenone-degradation gene has beenintroduced, thereby providing the ability to degrade or detoxifyzearalenone or a structurally related mycotoxin to said transformants.The present invention further provides a method for detoxification of aplant pre- or post-harvest using a microbe having the ability to degradeor detoxify zearalenone or structurally related mycotoxins. Theinvention also provides a method for detoxification of a plant pre- orpost-harvest using a zearalenone-degradation gene.

DISCLOSURE OF THE INVENTION

The present invention is based on the discovery of organisms with theability to degrade the mycotoxin zearalenone. The present invention hasresulted from a search for a biological means of detoxifyingzearalenones and comprises several bacterial species, isolated fromfield-grown maize kernels, and capable of growing on zearalenone as asole carbon source, degrading it partially or completely in the process.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of botany, microbiology, tissueculture, molecular biology, chemistry, biochemistry and recombinant DNAtechnology, which are within the skill of the art. Such techniques areexplained fully in the literature. See, e.g. J. H. Langenheim and K. V.Thimann, Botany: Plant Biology and Its Relation to Human Affairs (1982)John Wiley; Cell Culture and Somatic Cell Genetics of Plants, Vol. 1 (I.K. Vasil, ed. 1984); R. V. Stanier, J. L. Ingraham, M. L. Wheelis, andP. R. Painter, The Microbial World, (1986) 5th Ed., Prentice-Hall; O. D.Dhringra and J. B. Sinclair, Basic Plant Pathology Methods, (1985) CRCPress; Maniatis, Fritsch & Sambrook, Molecular Cloning: A LaboratoryManual (1982); DNA Cloning, Vols. I and II (D. N. Glover ed. 1985);Oligonucleotide Synthesis (M. J. Gait ed. 1984); Nucleic AcidHybridization (B. D. Hames & S. J. Higgins eds. 1984); the series inMethods in Enzymology (S. Colowick and N. Kaplan, eds., Academic Press,Inc.); and Current Protocols in Molecular Biology (John Wiley & Sons,Inc. 1996).

In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below.

A microbe is defined as any microorganism (including both eukaryotic andprokaryotic organisms) such as fungi, yeasts, bacteria, actinomycetes,algae and protozoa, as well as other unicellular structures capable ofgrowth in culture.

A zearalenone-producing microbe is any microbe capable of producing themycotoxin zearalenone or analogs thereof. Such microbes are generallymembers of the fungal genus Fusarium, as well as recombinantly derivedorganisms which have been genetically altered to enable them to producezearalenone or analogues thereof.

By zearalenone degradation, degrading zearalenone or having the abilityto degrade zearalenone is meant any modification or ability to make anymodification to the zearalenone molecule or a structurally relatedmycotoxin which causes a decrease in or loss of its toxic activity. Sucha change can comprise cleavage of any of the various bonds, oxidation,reduction, the addition or deletion of a chemical moiety, or any otherchange that affects the activity of the molecule. Furthermore,chemically altered zearalenone may be isolated from cultures of microbesthat produce an enzyme of this invention, such as by growing theorganisms on media containing radioactively-labeled zearalenone, tracingthe label, and isolating the degraded toxin for further study. Thedegraded zearalenone may be compared to the active compound for itsphytotoxicity or mammalian toxicity in known sensitive species, such asporcines.

By structurally related mycotoxin is meant any mycotoxin having achemical structure related to a zearalenone or analog of zearalenone, aswell as other a mycotoxin having a similar chemical structure that wouldbe expected to be detoxified by activity of the zearalenone-degradativeenzymes.

Harvested grain is defined as any form of grain which has been somehowremoved from the environment in which it was grown. For example,harvested grain may comprise ear corn, or corn kernels, for example.Harvested grain may further comprise that in storage or that beingprocessed. Processed grain is grain that has been through some form ofprocessing and will be used in the production of food for humanconsumption or will be used as animal feed ("feed grain").

Within this application, plant refers to a photosynthetic organismincluding but not limited to an algae, moss, fern, gymnosperm, orangiosperm. Preferably, said plant is one from which feed grain(preferably for human or animal consumption) may be harvested("harvested grain"). Most preferably, said plant includes any variety ofcorn (maize), wheat, sorgum, rice and barley.

A mature plant is defined as a plant in which normal development of allvegetative and reproductive organs has occurred.

A plant cell includes any cell derived from a plant, including callus aswell as protoplasts, and embryonic and gametic cells.

A regenerable culture is defined as a cell or tissue culture that can bemanipulated so as to allow regeneration of a plant.

A plantlet is defined as a plant sufficiently developed to have a shootand a root that is asexually reproduced by cell culture.

Explant refers to a section or piece of tissue from any part of a plantfor culturing.

The term callus and its plural calli refer to an unorganized group ofcells formed in response to cutting, severing or other injury inflictedon plant tissue. Excised pieces of plant tissue and isolated cells canbe induced to form callus under the appropriate culture conditions.Callus can be maintained in culture for a considerable time bytransferring of subculturing parts of the callus to fresh medium atregular intervals. The transfer of callus to liquid medium leads todispersion of the tissue and the formation of a plant cell suspensionculture. Callus can be induced to undergo organized development to formshoots roots.

Embryoid is defined as a structure similar in appearance to a plantzygotic embryo.

Somatic hybrid and somatic hybridization are generally defined as stablecombination of cellular material, be it protoplast/protoplast orprotoplast/cytoplast combinations, and includes cybrids andcybridization.

A transgenic plant is defined as any plant or plant cell that has becometransformed by the introduction, stable and heritable incorporation,into the subject plant or plant cell, of foreign DNA, i.e. DNA encodinga protein not normally found within that plant species.

A hormone is defined as any plant growth regulator that affects thegrowth of differentiation of plant cells. Such hormones includedcytokinins, auxins and gibberellins, as well as other substances capableof affecting plant cells.

A gene product that confers a selective advantage to a plant is definedas any gene product which, upon expression in said plant, confersincreased growth rate, yield of product or resistance to threats to saidplant's ability to thrive including but not limited to a pathogen, pest,adverse weather condition, and herbicide relative to a plant that doesnot express said gene product.

A replicon is any genetic element (e.g., plasmid, chromosome, virus)that functions as an autonomous unit of DNA replication in vivo; i.e.,capable of replication under its own control.

A vector is a replicon, such as a plasmid, phage, or cosmid to whichantoher DNA segment may be attached so as to bring about the replicationof the attached segment.

The term nucleotide sequence is defined as a DNA or RNA molecule orsequence, and can include, for example, a cDNA, genomic DNA, or asynthetic DNA.

A DNA fragment is defined as segment of a single- or double-stranded DNAderived from any source.

A DNA construct is defined a plasmid, virus, autonomously replicatingsequence, phage or linear segment of a single- or double-stranded DNA orRNA derived from any source.

A heterologous region of a DNA construct is defined herein as anidentifiable segment of DNA within or attached to another DNA moleculethat is not found in association with the other molecule in nature.Thus, when the heterologous region encodes a bacterial gene, the genewill usually be flanked by DNA that does not flank the bacterial gene inthe genome of the source bacterium. Another example of a heterologouscoding sequence is a construct where the coding seuqence itself is notfound in nature (e.g., synthetic sequences having codons different fromthe native gene). Heterologous DNA also refers to DNA not found withinthe host cell in nature. Allelic variation or naturally occurringmutational events do not give rise to a heterologous region of DNA, asthese terms are used herein.

The term polypeptide as used herein is used in its broadest sense, i.e.,any polymer of amino acids (dipeptide or greater) linked through peptidebonds. Thus, the term polypeptide includes proteins, oligopeptides,protein fragments, analogues, muteins, fusion proteins and the like. Theterm also encompasses amino acid polymers as described above thatinclude additional non-amino acid moieties. Thus, the term polypeptideincludes glycoproteins, lipoproteins, phosphoproteins, metalloproteins,nucleoproteins, as well as other conjugated proteins. The termpolypeptide contemplates polypeptides as defined above that arerecombinantly produced, isolated from an appropriate source orsynthesized.

A transcriptional regulatory region is defined as any element involvedin regulating transcription of a gene, including but not limited topromoters, enhancers and repressors.

A gene promoter is defined as any element involved in regulatingtranscription of a gene, including but not limited to promoters,enhancers and repressors.

A gene expressed in a tissue-preferred manner is that which demonstratesa greater amount of expression in one tissue as opposed to one or moresecond tissues in a plant specimen.

The term operably linked refers to the combination of a first nucleicacid fragment representing a transcriptional control region havingactivity in a cell joined to a second nucleic acid fragment encoding areporter or effector gene such that expression of said reporter oreffector gene is influenced by the presence of said transcriptionalcontrol region.

An assayable product includes any product encoded by a gene that isdetectable using an assay. Furthermore, the detection and quantitationof said assayable product is anticipated to be directly proportional tothe level of expression of said gene.

A reporter construct is defined as a subchromosomal and purified DNAmolecule comprising a gene encoding an assayable product.

An expression vector is defined as a subchromosomal and purified DNAmolecule comprising a transcriptional regulatory region drivingexpression of a gene.

An effector gene is defined as any gene that, upon expression of thepolypeptide encoded by said gene, confers an effect on an organism,tissue or cell.

Transformation refers to a method of introduction of DNA into a cell.Said introduction may include but is not limited to particlebombardment, lipofection, electroporation, viral or bacterialvector-mediated, and calcium phosphate mediated techniques.

The present invention comprises a methodology for the isolation of amicroorganism having the ability to degrade zearalenone or astructurally related mycotoxin, isolation of a gene encoding a geneproduct having the ability to degrade zearalenone, a methodology fordegradation of zearalenone or a structurally related mycotoxin on aplant in the field or post-harvest, a transgenic plant having theability to degrade zearalenone and a method for generating saidtransgenic plant. Said microorganism may include but is not limited tobacteria and fungi.

In order to isolate said microorganism having the ability to degradezearalenone or a structurally related mycotoxin, an assay was developedin which a microorganism is initially isolated from a source material.Said source material may comprise any plant or plant-associated materialincluding but not limited to any green tissue such as the stalk, leaf,ear, kernel, or soil in close approximation to the plant. To identify amicroorganism having the ability to degrade zearalenone, saidmicroorganism may be cultured in media containing zearalenone as thesole carbon source. Zearalenone, following addition to media, isgenerally found in a crystalline form. As zearalenone is degraded bysaid microorganism, the zearalenone crystals disappear from said media.The assay is termed a "crystal disappearance" assay. Degradation ofzearalenone may be confirmed using techniques including but not limitedto thin layer chromatography.

An important utility for the present invention is the detoxification ofzearalenone present upon or within a plant or grain following harvest. Asuitable feed material or "sample", that may include but is not limitedto cracked corn, chicken feed or corn meal, is spiked with a knownamount of mycotoxin delivered in a suitable solvent, preferably ethanol,at an appropriate rate, preferably one ml solvent per gram, followed bysufficient mixing to distribute said mycotoxin throughout said material.A control sample receives solvent only. The final concentration of saidmycotoxin is preferably between 0.1 and 1.0 mg per gram of feedmaterial. The sample may then be air-dried to remove excess solvent. Thesample is next inoculated with 10⁵ -10⁷ colony forming untis (cfu)/g oflog-phase cells of a microorganism having the ability to degrade saidmycotoxin, at a sufficient rate, preferably one ml cells per gram,followed by sufficient mixing to distribute said cells throughout saidsample. A control sample may comprise cells that have been killed byheating, preferably to approximately 80° C. A control sample may furthercomprise cells of a microorganism that is not able to degrade saidmycotoxin. Said sample is then placed into a container, said containeris closed and incubated for a sufficient period of time at anappropriate temperature. Said period of time is preferably within therange of one day to two weeks and said temperature is preferably roomtemperature or approximately 28° C. Following incubation, the contentsof said container is extracted in a suitable organic solvent (or organicaqueous mixture) for recovering said mycotoxin. The resulting extract isthen concentrated and subjected to qualitative and quantitative analysisfor the presence of said mycotoxin. The amount of said mycotoxindetected in said extract is then compared to the amount of saidmycotoxin detected in said control sample, and the efficacy of removalof said mycotoxin expressed as a percent reduction in the level of saidmycotoxin in said experimental extract as compared to the level of saidmycotoxin in said control sample. In the instant invention, saidmycotoxin is preferably zearalenone. This methodology allows for thedegradation of zearalenone upon or within said harvested plant or grain,thus providing improved food grain quality and feed safety.

Another important utility for the present invention is thedetoxification of zearalenone within or upon a plant in the field. Aplant may be inoculated with a zearalenone-producing organism and thentreated with an appropriate amount of bacteria having the ability todegrade zearalenone. The treatment may comprise application of acomposition comprising an efficacious amount of an organism having theability to degrade zearalenone to said plant whereby the zearalenonepresent is degraded. Preferably, said application consists of topicallyapplying said composition upon the tissues of said plant, such thatzearalenone upon said tissues is degraded. To generate a plant havingthe de novo ability to degrade zearalenone, a gene (the "gene ofinterest") encoding a gene product having the ability to degradezearalenone may isolated from said organism having the ability todegrade zearalenone and utilized to generate a transgenic plant.

It is possible to utilize any of several widely-available methodologieswell known to one skilled in the art to isolate the gene of interest andthe techniques described herein are not meant to limit the presentinvention to any certain methodology. One such method involves theisolation of a microorganism having the ability to degrade zearalenone(a "degrader"), isolation and limited cleavage of genomic DNA from saidmicroorganism into DNA fragments using a restriction enzyme, andtransformation of said DNA into a microorganism lacking the ability todegrade zearalenone (a "non-degrader"). Provided the gene of interest isincluded within a particular DNA fragment, transfer of said fragmentinto a non-degrader will confer upon said non-degrader a "degraderphenotype" (defined herein as the ability to degrade zearalenone). Thus,a fragment of DNA comprising a gene encoding a gene product having theability to degrade zearalenone may be identified. Said fragment may thenbe further digested into subfragments which may then be transformed intoa non-degrader organism. In this manner, the subfragment comprising saidgene of interest may be isolated. This cycle may be repeated to furtherlocalize said gene of interest. DNA sequence analysis of saidsubfragment may then identify a potential candidate for azearalenone-degradation gene. The potential candidate may then betransformed into a non-degrader and assayed for the ability to conferupon said non-degrader the degrader phenotype. In this manner, a geneencoding a gene product having the ability to degrade zearalenone may beisolated and identified.

Another method that may be utilized to isolate said gene of interest ispolymerase chain reaction (PCR)-based differential display analysis(Liang, et al. Science 257:967). This methodology involves the use ofrandom oligonucleotide primers, PCR-amplification of reversetranscriptase (RT)-cDNA and comparison of patterns of expression betweena first sample (preferably a degrader) and a second sample (preferably anon-degrader). Non-identical DNA banding patterns of DNA amplified fromsaid samples indicate a difference in gene expression between samples. ADNA fragment corresponding to a band that exhibits said non-identicalDNA banding pattern may then be isolated and utilized to isolate andidentify a gene to which said DNA band corresponds. The technique forisolation and identification of said gene is widely available to oneskilled in the art.

Another method for isolating said gene of interest is cloning bysubtractive hybridization (Lee, et al. Proc. Natl. Acad. Sci. 88:2825).Antisense cDNA of sample A (preferably a non-degrader) andbiotinylated-RNA of sample B (preferably a degrader) are hybridized.Biotinylated-RNA molecules of the degrader organism representing genesexpressed in both the non-degrader organism and the degrader organismwill hybridize to the complementary cDNA molecules of the non-degraderto form hybrids. Said hybrids are then destroyed by enzymatic treatment.The remaining biotinylated RNA molecules of the degrader organismrepresent those genes expressed at a higher level in said degrader thanin said non-degrader. Said biotinylated RNA molecules are then purifiedand a cDNA library representing said remaining biotinylated RNA of saiddegrader is constructed. The genetic material within said cDNA librarymay represent genes that are preferentially expressed in the degraderorganism.

A further method comprises screening of a cDNA library of a first sample(preferably a degrader organism) using labeled RNA representing a secondsample (preferably a non-degrader organism). Genetic material of saidcDNA library of said first sample that does not hybridize to saidlabeled RNA of said second sample may represent RNA species that are notexpressed in said second sample. Said genetic material may then betransformed into a non-degrader organism and said organism is assayedfor the ability to degrade zearalenone using as assay such as thecrystal disappearance assay. The ability of said genetic material toconfer upon a non-degrader organism said degrader phenotype indicatesthat said genetic material comprises a gene encoding a gene producthaving the ability to degrade zearalenone (a gene of interest).

A further method that may be utilized to isolate a gene encoding a geneproduct capable of metabolizing zearalenone comprises purification ofthe gene product using conventional protein purification techniques.Many different techniques are available to one skilled in the art forpurification of said gene product, and the techniques described hereinare not meant to limit the present invention to a certain methodology.An extract is prepared from a bacterial isolate known to have theability to degrade or detoxify zearalenone. The extract is preparedusing conditions that maintain the ability of the protein of interest todegrade zearalenone. Fractions of the extract are then separated basedon a certain biochemical property or combination of properties of theproteins comprising said fractions. Such properties allow separation ofproteins based on characteristics including but not limited to molecularsize, charge, and conformation using techniques including but notlimited to size-exclusion chromatography, ion exchange chromatography,reverse phase chromatography, precipitation, centrifugation, andelectrophoresis. Proteins of the extracts that share certain propertiesare collected into fractions. Each of said fractions is then assayed forthe ability to degrade zearalenone, allowing for identification offractions comprising a protein having the ability to degradezearalenone. This fraction may then be further separated intosubfractions using any of the above-described or any other availabletechniques. The proteins comprising said subfractions may then beassayed for the ability to degrade zearalenone. Said subfractions may befurther fractionated (ultimately providing a partially or completelypurified protein) in order to increase the purity of the protein havingthe ability to degrade zearalenone.

Following partial or complete purification of a protein having theability to degrade zearalenone, a probe useful in identifying the geneencoding said protein is generated. Said probe may comprise anoligonucleotide or an antibody. In order to generate an oligonucleotideprobe, the partially or completely purified protein havingzearalenone-metabolizing activity is subjected to partial or completeamino acid sequence determination using techniques widely available toone skilled in the art, including but not limited to Edman degradation.The partial amino acid sequence is determined and an oligonucleotide isdesigned based on the amino acid sequence determination. The methodologyfor designing an oligonucleotide based on amino acid sequence is widelyavailable and well known to those skilled in the art. Theoligonucleotide comprises a region substantially identical to codonswhich encode the amino acid sequence of said protein having the abilityto degrade zearalenone. Said oligonucleotide probe may be utilized toscreen a nucleic acid library representing the genetic material of anorganism having the ability to degrade zearalenone. Multiple distinctoligonucleotides may be designed and utilized for screening said nucleicacid library in order to perform multiple sequential hybridizations tolimit isolation of "false positive" samples. The methodology forscreening genomic DNA libraries is well known to those skilled in theart. Alternatively, several of said oligonucleotides may be designed foruse in PCR-mediated cloning of a gene encoding a gene product having theability to degrade zearalenone. PCR amplification of genetic material isa well-known method widely available to one skilled in the art. Saidoligonucleotides may be utilized to amplify genetic material comprisinga gene encoding a gene product having the ability to degradezearalenone. Said genetic material may be isolated from a degraderorganism and subjected to PCR amplification using said oligonucleotidesas primers.

An antibody probe may be generated and utilized to isolate the gene thegene of interest. The above-described partially or completely purifiedprotein product having the ability to degrade zearalenone may beutilized to immunize an animal host resulting in the generation of anantibody that binds to said protein. Methodologies involved inimmunizing an animal with a partially or completely purified proteinpreparation and isolating the resulting antibody are widely availableand well known in the art. The animal host may include but is notlimited to a rabbit, mouse, rat, hamster, or guinea pig. Said antibodymay comprise a whole antibody or a fragment thereof. An antibody orantibody fragment may also be generated using a phage display system.This method may comprise the use of an affinity column onto which isadsorbed the purified protein having zearalenone-metabolizing activityor a fragment thereof. A phage capsid comprising an antibody or antibodyfragment capable of binding to said protein is applied to said columnresulting in the interaction of a ligand (preferably said purifiedprotein) and said antibody or antibody fragment on the surface of saidphage. The gene encoding said antibody or antibody fragment may then beisolated from said phage and utilized to generate sufficient quantitiesof said antibody or antibody fragment to be utilized as an antibodyprobe. Said antibody probe may then be tested for binding to theproteins within the fraction containing the partially or completelypurified protein having zearalenone-metabolizing activity.

A method that may be utilized to test the specificity of the antibodypreparation includes but is not limited to western blot, a well knownmethodology well known to one skilled in the art. Proteins within saidfraction separated electrophoretically using a technique such as gelelectrophoresis which is well known and widely available to one skilledin the art. Following separation of said proteins of said fraction, saidproteins are transferred to a solid support such as nitrocellulose orPVDF membrane which is then probed with said antibody preparation.

Provided said antibody binds to a protein bound to said solid support,said antibody may be detected using any of several antibody detectiontechniques that are well known and widely available to those skilled inthe art. One such detection method includes the use of a labeledsecondary antibody that demonstrates reactivity against said antibody.Said secondary antibody may be coupled to a detectable probe such as aradioactive nucleotide, an enzyme or biotin. Upon binding of the labeledsecondary antibody to the primary antibody, the labeled secondaryantibody may be detected using any of several widely available detectionmethodologies. One such detection method is the Enhanced ChemluminescentDetection System available from Amersham Corp.

A further method for testing said antibody for reactivity against saidprotein is the ELISA assay, the methodology of which is well known andwidely available to those skilled in the art. A sufficient amount ofsaid fraction is applied to a well of an assay plate, allowing theproteins within said fraction to bind to said well. A protein to whichthe antibody preparation react are then detected by application of saidantibody followed by detection of said antibody using the ELISAdetection methodology.

An antibody having reactivity to a protein within said fraction may thenbe utilized to screen an expression library comprising a gene foundwithin the genetic material of a degrader organism. Methods forisolation of clones from such a library using an antibody are well knownand widely available to those skilled in the art. Screening of saidgenomic library is accomplished by plating an organism transformed witha plasmid or infected with a bacteriophage comprising a portion of saidgenomic library. The genomic library is prepared such that thetransformed organism will express a protein encoded by a gene withinsaid genomic fragments. Said organisms or said genomic fragments of saidorganisms are then transferred to a solid support such asnitrocellulose, nylon or PVDF membrane and probed with said antibodyhaving reactivity to a protein or proteins within said fraction. Anorganism that expresses the protein or proteins of interest is detectedby reactivity with said antibody. Such an organism may then be isolatedand further screening performed to isolate a pure population of saidorganism carrying said genomic fragment encoding said protein orproteins of interest. Purification may require several cycles ofscreening. In this manner, it is possible to isolate a genomic fragmentcomprising genetic material comprising a gene encoding a gene producthaving the ability to degrade zearalenone.

Confirmation that said genetic material isolated by any of theabove-described techniques is expressed in a degrader organism may beachieved using any of several widely available techniques well known toone skilled in the art. A method that may be utilized to determine thelevel of gene expression is the RNase protection assay (Melton, et al.Nuc. Acids Res. 12:7035). RNA from the samples to be compared isisolated and hybridized to a labeled antisense RNA probe correspondingto the genetic material comprising a gene encoding a gene product havingthe ability to degrade zearalenone (Summers, 1970, Anal. Biochem.33:459; Thomas, 1980, PNAS 77:5201). This is followed by the addition ofRNase which will degrade non-hybridized transcripts. RNA that hashybridized to said RNA probe is protected from degradation (termedprotected transcripts) by the RNase while mRNA that has not hybridizedto said RNA probe is degraded. The products are then separated by gelelectrophoresis and protected transcripts detected using detectionmethods including but not limited to autoradiography. The relativeintensity of the band corresponding to said protected transcript isproportional to the level of expression of gene said transcriptrepresents in the organism.

An additional method by which the level of gene expression may bedetermined is the northern blot analysis (Alwine, et al. Proc. Natl.Acad. Sci. 74:5350). RNA from a sample is isolated and separated by gelelectrophoresis. The separated RNA species are then transferred to amembrane and probed with a labeled nucleic acid probe that iscomplementary to RNA representing a gene of interest. Hybridization isdetected using a detection method including but not limited toautoradiography. The intensity of the band corresponding to RNArepresenting a gene of interest is determined and is proportional to thelevel of gene expression in the sample. Preferably, one sample is adegrader organism and another sample is a non-degrader organism. Thelevel of gene expression of said gene of interest in the degraderorganism is preferably increased in said degrader organism as comparedto said non-degrader organism.

It may then be useful to construct an expression vector for testing theability of said genetic material to confer the ability to metabolizezearalenone upon a non-degrader organism following transformation withsaid gene. A transcriptional control region able to drive geneexpression in said organism may be linked in cis to said geneticmaterial. Said expression vector may then be transformed into anorganism that does not have the ability to degrade zearalenone.Following transformation, a transformed organism may be tested for theability to degrade zearalenone using an assay such as the crystaldisappearance assay. The ability of said non-degrader to degradezearalenone following transformation with said expression vectorindicates that a zearalenone gene has been isolated.

An expression vector comprising a transcriptional regulatory region thatdrives gene expression in plants operably linked to said geneticmaterial comprising a gene encoding a gene product having the ability todegrade zearalenone may also be constructed. Said expression vector maybe transformed into a plant cell or plant tissue. The method utilizedfor transformation of various types of plant cells or plant tissues maycomprise particle bombardment, liposome-mediated transformation, calciumphosphate-mediated transformation, bacterial- or viral-mediated genetransfer, electroporation, or Agrobacterium-mediated transformation. Aplant cell or plant tissue may be transformed in vitro after excisionfrom said plant. Following a defined period of time after transformationof said expression vector into said plant cell or plant tissue, saidplant cell or plant tissue may be harvested and an assay capable ofdetecting said gene product having the ability to degrade zearalenoneperformed. Said assay may comprise direct detection using an antibody orother probe or indirectly by measuring the ability of an extract derivedfrom said plant cell or plant tissue to degrade zearalenone.

A transgenic plant having a copy of said gene of interest incorporatedinto the genome of the plant may be generated. A regenerable culture ofa plant may be transformed with an expression vector comprising a geneencoding a gene product having the ability to degrade zearalenone. Themethod utilized for transformation of said regenerable culture maycomprise particle bombardment, liposome-mediated transfection, calciumphosphate-mediated transfection, bacterial- or viral-mediated genetransfer, electroporation, or Agrobacterium-mediated transformation.Following transformation, said regenerable culture may be regeneratedinto a mature transgenic plant. Harvest of a tissue from said transgenicplant may then be performed followed by assay of said tissue for thepresence of said gene product having the ability to degrade zearalenone.Said assay may comprise direct detection using an antibody or otherprobe or indirectly by measuring the ability of an extract derived fromsaid plant cell or plant tissue to degrade zearalenone.

Tests may be performed on said transgenic plant to determine the abilityof said transgenic plants to degrade zearalenone in the field. Saidtransgenic plant may inoculated at an early stage with azearalenone-producing organism. Said transfected plant may then beharvested and an inoculated portion assayed for the presence ofzearalenone.

A further test of the ability of said transgenic plant to degradezearalenone may comprise feeding of said transgenic plant or grainharvested from said transgenic plant to a test animal such as a pig.Zearalenone has been shown to incite adverse effects in pigs includingbut not limited to an estrogenic response including infertility, reducedlitter size and weak piglets. Zearalenone has also been shown to bephysiologically active in cattle, rats, mice, guinea pigs, and poultry,any of which may also be utilized as a test animal. Said transgenicplant may be inoculated with a zearalenone-producing organism and, atthe appropriate time, the inoculated transgenic plant or harvested grainfrom said transgenic plants may be fed to a test animal or animals. Asan experimental control, another animal or animals are fed anon-transgenic plant or harvested grain from said non-transgenic plantthat have been inoculated with a zearalenone-producing organism in anidentical manner to that of said transgenic plant. The test animal oranimals may then be observed for the presence of any adverse effectsknown to be associated with exposure to zearalenone. The presence ofsaid adverse effects in an animal fed said non-transgenic, inoculatedplant or harvested grains from said plant, and the lack of said adverseeffect in an animal fed said transgenic, inoculated plant or harvestedgrain of said plant indicates that expression of said gene encoding agene product having the ability to degrade zearalenone in saidtransgenic plant confers the ability to degrade zearalenone to saidplant.

This invention can be better understood by reference to the followingnon-limiting examples. It will be appreciated by those skilled in theart that other embodiments of the invention may be practiced withoutdeparting from the spirit and the scope of the invention as hereindisclosed and claimed.

EXAMPLE I. ISOLATION OF BACTERIA THAT DEGRADE ZEARALENONE

Various sources of plant material that were likely to naturally containzearalenone were collected as source material for screening. Wheatkernels (140 independent samples) infested with Fusarium graminearum,the causal agent of wheat scab, were obtained from a Pioneer Hi-BredInternational, Inc. ("Pioneer") wheat breeding station in Indiana.Silage samples were obtained from the Microbial Genetics division ofPioneer Hi-Bred and compost samples from local residences (139independent samples total). Fusarium graminearum-infested maize kernelswere obtained from a Pioneer Gibberella zeae (Fusarium graminearum)disease nursery (121 independent samples).

The metabolism of zearalenone was measured using the crystaldisappearance assay. Microbes were washed from the source material byplacing a small amount in a seven milliliter Falcon tube and adding oneto two milliliters sterile distilled water (producing "wash fluid").Maize kernels were split with a razor blade and one to two kernels wereused. Tubes were capped and shaken for one to three hours at roomtemperature. Zearalenone (Sigma Cat. No. Z0167) was prepared as asuspension in mineral salts medium, and was utilized as the sole carbonsource. The zearalenone concentration utilized includes but is notlimited to 0.75-1.0 milligrams/milliliter in mineral salts medium. Themineral salts medium was prepared by combination of reagents includingbut not limited to 1.0 g/L ammonium sulfate, 1.0 g/L sodium chloride,1.0 g/L potassium phosphate, dibasic, 0.2 g/L magnesium sulfate.Sterilization of the solution was accomplished by filtration through a0.2 micron filter, although various methods for sterilization areavailable to those skilled in the art. 100 microliters ofzearalenone/mineral salts suspension medium was added to each well of amicroliter plate (96 well plate). One microliter of wash fluid from saidsource material was added to each well. Control wells received onemicroliter of water. After two weeks, one microliter from each well wastransferred to a new microliter plate containing 100 microliters ofzearalenone/mineral salts medium. The transfer was then repeated fourweeks later. After six weeks, wells were scored for partialdisappearance of zearalenone crystals. Typically, the small crystals hadbeen solubilized and metabolized, and only the very largest zearalenonecrystals remained. This effect was visualized using an invertedmicroscope or by examining the plate visually from the underside.

Crystal disappearance was verified by thin layer chromatography (TLC).Silica gel plates containing fluorescent indicator (Whatman 4410 222)were spotted with typically one microgram zearalenone (typically onemicroliter from assay plates or one microliter of a onemiligram/milliliter standard solution). Plates were run using a solventsystem of chloroform-ethyl alcohol 97:3. Zearalenone could be seen as abright blue spot under short-wave UV. Microbial metabolism ofzearalenone caused a gradual disappearance of the spot; spots withaltered mobility in the TLC system were not detected using this method.

The instant invention comprises a biologically pure culture of amicroorganism having the ability to degrade zearalenone. Saidmicroorganism was isolated using the following procedure. One microliterwas removed from each well in which degradation of zearalenone wasobserved and added to one milliliter of sterile water. Several ten-folddilutions were made in sterile water, and 100 microliters from eachdilution were plated and spread on YDP agar plates. YDP agar plates wereprepared by combination of 10 grams yeast extract (Difco), 20 g Bactopeptone, 0.5 g dextrose, 15 g Bacto agar in water followed bysterilization by autoclaving. From these mixture culture spread plates,individual colonies were streaked for isolation on new YDP plates. Aneffort was made to choose at least one of every type of bacteriarepresented on the spread plates. Each bacterium was used to make adilute suspension in sterile water, and one microliter of thissuspension was used to inoculate microliter wells containing zearalenonein mineral salts as described above.

Initial characterization of bacteria was performed by Gram stainingsamples. More definitive identification was performed using acombination of techniques. Streak plates of individual bacterialcolonies were sent to Microbe Inotech Laboratories, Inc. (St. Louis,Mo.) for tentative identification. The analysis included comparison ofbacterial fatty acid methyl esters with Aerobe and Clinical Aerobedatabases, and Biolog™ substrate utilization comparison with a Grampositive database. Results of such tests indicate that the bacterialisolate is likely of the Rhodococcus or Nocardia species. From a totalof 386 samples, zearalenone-degrading bacteria were isolated from fourindependent sources of Fusarium graminearum-infested maize. The data issummarized below in Table 1. Each of the four samples were deposited onOct. 15, 1996 (ATCC 55853, ATCC 55852, ATCC 55851) and Oct. 22, 1996(ATCC 55856) with the American Type Culture Collection (ATCC; 12301Parklawn Drive, Rockville, Md. 20852 USA) in accordance with theBudapest Treaty on the International Recognition of the Deposit ofMicroorganisms for the Purposes of Patent Procedure.

                  TABLE 1    ______________________________________    Microbial isolates having the ability to degrade zearalenone    ATCC # Name        Tentative Identification                                       Source    ______________________________________    55856  2855.1      Rhodococcus globulerus or                                       Moldy corn,    Michigan           Rhodococcus erythropolis    55853  ZEA(1)2906.C1                       Nocardia globulera or                                       Moldy corn,    Michigan           Rhodococcus erythropolis    55852  ZEA(1)2906.C4                       Nocardia globulera or                                       Moldy corn,    Michigan           Rhodococcus erythropolis    55851  ZEA(1)2906.A8                       Rhodococcus erythropolis                                       Moldy corn,    Michigan    ______________________________________

EXAMPLE II. TREATMENT OF ZEAROLENONE-CONTAMINATED CORN A. Treatment ofContaminated Corn in the Field

To test the ability of the bacteria isolated by the above-describedmethodology to degrade or detoxify zearalenone or its derivatives oranalogs on maize, mature plants are inoculated with azearalenone-producing Fusarium sp. and then treated with an appropriateamount of bacteria having the ability to degrade or detoxify zearalenoneor its derivatives or analogs. The treatment consists of topicallyapplying a composition comprising an efficacious amount of bacteria ontothe tissues of the maize plant such that zearalenone, potentiallyincluding any derivatives or analogs of zearalenone, is partially orcompletely degraded or detoxified.

B. Treatment of Contaminated Corn after Harvest.

A one to ten gram sample of cracked corn is combined or "spiked" with aknown amount of zearalenone in ethanol at a concentration of one gramzearalenone per ml of ethanol, followed by mixing to distribute thezearalenone throughout the mixture. A control sample or samples aremixed with solvent alone. The samples are then air-dried to removeexcess solvent. The samples are then inoculated with 10⁶ cfu/g oflog-phase cells of a microorganism having the ability to degradezearalenone, designated 2855.1 (deposited with the ATCC under accessionnumber ATCC 55856) at a rate of one ml cells per gram, and mixed well todistribute said cells within said sample. Controls are mixed with eithercells of said microorganism (designated 2855.1, deposited with the ATCCunder ATCC accession number 55856) that have been heated to 80° C., suchthat said cells are non-viable or with cells of a microorganism thatdoes not have the ability to degrade zearalenone. Said mixture is placedin a container, which is then closed and incubated for two weeks at roomtemperature. At the end of the incubation period, the container isopened, and the entire contents extracted in a suitable organic solventto recover the zearalenone. The extract is concentrated and subjected toqualitative and quantitative analysis for detection of zearalenone. Theamount of zearalenone is determined and compared to controls. Theefficacy of removal of zearalenone is determined by comparison of thepercent reduction of the amount of zearalenone in the sample comprisingthe micoorganism having the ability to degrade zearalenone to thereduction (if any) of the amount of zearalenone present in said controlsample. Microorganisms designated ZEA(1)2906C.1 (ATCC accession number55853), ZEA(1)2906.C4 (ATCC accession number 55852), or ZEA(1)2906.A8(ATCC accession number 55851) are also able to degrade zearalenone, andmay be utilized for the above-described purpose.

EXAMPLE III. CLONING OF A ZEARALENONE-DEGRADATION GENE

Following isolation of a microorganism having the ability to degradezearalenone (a "degrader"), the genomic DNA of said microorganism isisolated and treated with a restriction enzyme into DNA fragments. SaidDNA fragments are then cloned into an expression vector having atranscriptional control region able to drive gene expression inbacteria. Said expression vector comprising said transcriptional controlregion and a DNA fragment of said degrader organism is then transformedinto a bacteria lacking the ability to degrade zearalenone (a"non-degrader"). Following transformation, the non-degrader bacteria aretested for the ability to degrade zearalenone (defined as the "degraderphenotype").

The metabolism of zearalenone is measured using a crystal disappearanceassay. Zearalenone (Sigma Cat. No. Z0167) is prepared as a suspension inmineral salts medium, and utilized as the sole carbon source. Thezearalenone concentration is 0.75-1.0 milligrams/milliliter in mineralsalts medium. The mineral salts medium is prepared by combination of 1.0g/L ammonium sulfate, 1.0 g/L sodium chloride, 1.0 g/L potassiumphosphate, dibasic, 0.2 g/L magnesium sulfate. Sterilization of thesolution is accomplished by filtration through a 0.2 micron filter. 100microliters of zearalenone/mineral salts suspension medium is added toeach well of a microliter plate (96 well plate) containing onemicroliter of a media containing said non-degrader bacteria. After twoweeks, one microliter from each well is transferred to a new microliterplate containing 100 microliters of zearalenone/mineral salts medium.The transfer is then repeated four weeks later. After six weeks, wellsare scored for partial disappearance of zearalenone crystals. Thiseffect is visualized using an inverted microscope or by examining theplate visually from the underside. Crystal disappearance is verified bythin layer chromatography (TLC). Silica gel plates containingfluorescent indicator (Whatman 4410 222) are spotted with one microgramzearalenone. Plates are run using a solvent system of chloroform-ethylalcohol 97:3. Microbial metabolism of zearalenone causes a gradualdisappearance of the spot; spots with altered mobility in the TLC systemare not detected using this method.

Provided the gene of interest is included within a particular DNAfragment, transfer of said fragment into a non-degrader confers uponsaid non-degrader said "degrader phenotype". Thus, a fragment of DNAcomprising a gene encoding a gene product having the ability to degradezearalenone is identified. Said fragment is then subjected to DNAsequencing in order to identify the genes included within said fragment.Following identification of a gene encoding a gene product having theability to degrade zearalenone within said fragment, said gene isisolated and an expression vector comprising said gene is constructed.Said expression vector is then transformed into non-degrader bacteria,and said bacteria are isolated and tested for expression of the degraderphenotype. The presence of said degrader phenotype in a transformednon-degrader indicates that said gene encodes a gene product having theability to degrade zearalenone.

EXAMPLE IV. TRANSGENIC CORN HAVING ZEAROLENONE-DEGRADING ACTIVITY

To provide a maize plant having the ability to degrade zearalenone, atransgenic maize plant is generated by transformation of azearalenone-degradation gene into a maize regenerable culture(preferably derived from maize that do not have the ability to degradezearalenone) followed by regeneration of said regenerable culture into amature transgenic maize plant. Said maize regenerable culture istransformed by particle bombardment with an expression vector comprisinga zearalenone-degradation gene. Following regeneration of said maturemaize plant, certain tissues of said transgenic plant are harvested andan assay capable of detecting the zearalenone-degradation gene productis performed. Said gene product is detected by measuring thezearalenone-metabolizing activity of transfected cell extracts or tissueextracts from transfected tissue segments by TLC using radiolabeledzearalenone.

The ability of the zearalenone-degradation gene to confer the degraderphenotype upon said transgenic maize is tested. The developing ears ofsaid transgenic maize are inoculated at the early silking stage usingtoothpicks impregnated with Fusarium graminearum. The infected corn cobsare then machine harvested and the infected portions of the cob andkernels assayed for the presence of zearalenone. The ability of saidgene to confer the ability to degrade zearalenone upon said transgenicmaize is indicated by the lack of zearalenone in tissues of saidtransgenic maize. Non-transgenic maize are inoculated in an identicalmanner as said transgenic maize and zearalenone is detected in saidnon-transgenic maize.

A further test of the ability of the transgene to providezearalenone-degradation activity to transformed maize plants includesfeeding the transgenic maize to animals such as pigs. Said transgenicmaize is inoculated with Fusarium graminearum and, at the appropriatetime, the inoculated transgenic corn is harvested and fed to the testanimals. As an experimental control, certain other animals are fednon-transgenic corn that has been inoculated in an identical manner tothat of said transgenic maize and, at the appropriate time, said maizeis harvested and fed to the test animals. The animals are studied forthe presence of any of the above-described estrogenic effects ofzearalenone including infertility, reduced litter size and weak pigletsafter eating the Fusarium-inoculated transgenic maize or theFusarium-inoculated non-transgenic maize. While the presence of anestrogenic response is observed in animals fed said Fusarium-inoculated,non-transgenic maize, an estrogenic response is not observed in animalsfed said Fusarium-inoculated, transgenic maize. The absence of theestrogenic response in animals fed said Fusarium-inoculated, transgenicmaize indicates that expression of said zearalenone-degradation gene insaid transgenic maize provides said transgenic maize with the ability todegrade zearalenone to a non-toxic level in and on tissues of saidtransgenic maize.

While a preferred form of the invention has been shown in the drawingsand described, since variations in the preferred form will be apparentto those skilled in the art, the invention should not be construed aslimited to the specific form shown and described, but instead is as setforth in the claims.

What is claimed is:
 1. An isolated and biologically pure bacteriumhaving the ability to degrade zearalenone wherein said bacterium is ofthe Rhodococcus species or the Nocardia species.
 2. The bacterium ofclaim 1 wherein said bacterium is selected from the group consisting ofRhodococcus globerulus, Rhodococcus erythropolis, and Nocardiagloberula.
 3. The bacterium of claim 2 wherein said bacterium isselected from the group consisting of the bacterium deposited under ATCCaccession number 55856, the bacterium deposited under ATCC accessionnumber 55853, the bacterium deposited under ATCC accession number 55852,and the bacterium deposited under ATCC accession number 55851.